Methods for multi-dose synthesis of [f-18]fddnp for clinical settings

ABSTRACT

A method of manufacturing 2-(1-{6-[(2-[F-18]fluoroethyl)(methyl)amino]-2-naphthyl}ethylidene)-malononitrile ([F-18]FDDNP) utilizes a semi-automated module that is used to perform fluorination, pre-purification, separation, product extraction, and formulation. The method is able to produce [F-18]FDDNP with high yields and ready for human administration under existing FDA regulations, and without the need for hazardous organic solvents such as dichloromethane (DCM), methanol (MeOH), and tetrahydrofuran (THF). The method also improves the speed with which [F-18]FDDNP can be synthesized with the method being able to generate a final product within about 90 to 100 minutes. This synthesis method is easily adaptable to FDA registered and approved automated synthesis systems.

RELATED APPLICATION

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/419,286 filed on Nov. 8, 2016, which is hereby incorporated byreference in its entirety. Priority is claimed pursuant to 35 U.S.C. §119 and any other applicable statute.

TECHNICAL FIELD

The technical field generally relates to a method of synthesizing2-(1-{6-[(2-[F-18]fluoroethyl)(methyl)amino]-2-naphthyl}ethylene)-malononitrile([F-18]FDDNP) using a semi-automated synthesis module. The method isused to synthesize [F-18]FDDNP with high yields and short productiontimes, ready for human administration. The method also introduces anon-aqueous workup procedure and does not use a combination of hazardousorganic solvents that have been used in prior synthesis operations.

BACKGROUND

2-(1-{6-[(2-[F-18]fluoroethyl)(methyl)amino]-2-naphthyl}ethylidene)-malononitrile([F-18]FDDNP) PET imaging has been used for classifying and stagingprogressive diseases, like Alzheimer's disease (AD) and ChronicTraumatic Encephalopathy (CTE). Almost two decades of clinical researchexperience in the U.S., Europe, and Asia has demonstrated the ability of[F-18]FDDNP to differentiate Alzheimer's disease (AD) from normal aging,mild cognitive impairment, and several other neurodegenerative diseases(e.g., progressive supranuclear palsy, dementia with Lewy bodies, Downsyndrome). The ability of [F-18]FDDNP to differentiate AD from normalaging is comparable to that of 2-deoxy-2-[F-18]fluoro-D-glucose([F-18]FDG). Moreover recent clinical research demonstrates a distinct[F-18]FDDNP binding pattern in retired athletes and military personnelwith a history of traumatic brain injury and suspected CTE, and thispattern can be readily differentiated from that of AD. Currently thereis no available biomarker that can detect suspected CTE in living peopleat risk, and other PET ligands for this purpose are very early in theirdevelopment.

Liu et al. discloses one method for the automated radiosynthesis of[F-18]FDDNP. See Liu, J. et al., High-yield, automated radiosynthesis of2-(1-{6-[(2-[¹⁸F]fluoroethyl)(methyl)amino]-2-naphthyl}ethylidene-malononitrile([¹⁸F]FDDNP)ready for animal or human administration, Mol. Imaging Biol., 9: 6-16(2007). However, this previously reported synthesis of [F-18]FDDNP forthe preparation of multi-dose quantities of tracer has certainlimitations. First, this method uses a rather complex pre-purificationof the F-18 fluorination reaction mixture using cumbersome multiplecartridges and evaporation processes prior to semi-preparative HPLCpurification. Second, it uses toxic and potentially harmful organicsolvents in the pre-purification and semi-preparative HPLC purificationsteps (e.g., dichloromethane, methanol (MeOH), and tetrahydrofuran).Third, autoradiolytic decomposition of [F-18]FDDNP could potentiallyoccur during the pre-purification and HPLC purification processes.Autoradiolytic decomposition can generally be a serious issue with F-18labeled compounds with high specific activities leading to a reductionin radiochemical yields, decreased product stability, and increase inradiochemical impurities. In the previously reported synthesis, theformulation and sterilization of the product in human serum albumin(HSA) also lowers the radiochemical yield of the final product to about27%. Radiochemical yields are particularly important because positronemitter labeled biomarkers like [F-18]FDDNP have a relatively shorthalf-life (half-life of F-18 isotope is 110 minutes).

Two considerations are important for the use of these biomarkers inclinical settings, which is the primary intent for the use of[F-18]FDDNP to characterize CTE and AD, among other neurodegenerativediseases. First, such biomarkers or probes are often produced in onegeographic location (e.g., where a cyclotron is located), but used inremote geographic regions. Transportation of F-18 labeled biomarkersover relatively longer geographical distances will invariably result inlower final usable doses. It is thus critically important to achieve ashigh a radiochemical yield as possible so that one batch of [F-18]FDDNPcan be divided into multiple doses that can efficiently be used even ingeographically remote locations. Second, in U.S. Food and DrugAdministration (FDA)-sponsored clinical trials and clinical use of PETbiomarkers, the easiness, reproducibility and reliability of the finalproduct ready for injection are essential.

SUMMARY

A modular method for the synthesis of [F-18]FDDNP is disclosed forclinical settings. This approach is highly reliable and can produce upto one hundred (100) 10 mCi batch doses of pure, high specific activity(typically from 1 to 5 Ci/micromole) [F-18]FDDNP biomarker ready forhuman injection using available biomedical cyclotrons routinelyproducing up to 5-10 Ci of [F-18]fluoride ion. Product preparation bytrained technicians using this method is thus easily achievable, animportant condition in high throughput clinical settings. Alternatively,the synthesis method may be implemented in an automatedradiosynthesizer. The method reduces synthesis time, significantlysimplifies the synthesis procedure, most specifically the complexpre-purification of the F-18 fluorination reaction mixture usingcumbersome multiple cartridges and organic solvents, and evaporationprocesses prior to semi-preparative HPLC purification. Among others, acritical improvement made in the method includes utilization of analumina-based cartridge for the purification of the crude radioactivereaction mixture and inclusion of ascorbic acid in the semi-preparativeHPLC mobile phase. A main purpose of the synthesis procedure disclosedherein is to have a high throughput chemical synthesis, a reliablechemical method which essentially covers two purposes: (1) a high yieldsynthesis, which would permit transportation of a pure, final product torelatively distant sites to compensate for product decay (at least tosome extent) due to the short half-life of the F-18 isotope (half-life:110 min); and (2) the ease of synthesis will permit easy adaptation ofthis synthesis to any FDA-approved automated radiosynthesizer (ormanual/semi-automated synthesis setups) at any sites having cyclotrons.Thus, the utilization of this PET probe could be greatly facilitated atmultiple clinical sites. The synthesis method for [F-18]FDDNP disclosedherein is easily adaptable to most FDA-approved automatic synthesisdevices for PET biomarkers.

The use of the alumina cartridge was found to be superior to theincommodious multiple cartridge system previously utilized in theinitial purification of the F-18 fluorinated reaction mixture. In themethod used herein, a single alumina cartridge efficiently removes, in asingle step, inorganic materials (e.g., K₂CO₃) and cryptands (e.g.,Kryptofix®), as well as some of the organic side products formed duringthe F-18 fluorination reaction, while causing less decomposition. Theinclusion of ascorbic acid in the semi-preparative HPLC mobile phase hada beneficial effect particularly when using aqueous acetonitrile in thepurification process. For example, ascorbic acid prevented theautoradiolytic decomposition of [F-18]FDDNP during the HPLC purificationprocess.

In one embodiment, no-carrier-added [F-18]fluoride ion was produced by11 MeV proton bombardment of 90-98% enriched [O-18]water. The aqueous[F-18]fluoride ion was trapped on an anion resin cartridge to recoverthe [O-18]water. The [F-18]fluoride ion was subsequently released bypassing 0.25% (weight:volume basis) aqueous solution (0.4 mL) of apotassium salt (e.g., K₂CO₃) into a glass reaction vessel pre-loadedwith 4,7,13,16,21,24-Hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane cryptand(e.g., Kryptofix® 2.2.2.) (10 mg) dissolved in 1.0 mL of acetonitrile.The solution was evaporated at 115° C. with a stream of nitrogen gasbubbling into it. The residue was dried by the azeotropic distillationwith acetonitrile (3×0.5 mL). To the dry residue, a solution of thetosyloxy precursor2-{[6-(2,2-dicyano-1-methylvinyl)-2-naphthyl](methyl)amino}ethyl-4-methylbenzenesulfonate(DDNPTs) (2.2-2.5 mg) in acetonitrile (0.7 mL) was added and thereaction mixture was heated at 93° C. for 15 min. The solution wascooled with a nitrogen stream for 1 min. The mixture was then passedthrough a neutral alumina-containing cartridge (e.g., Sep-Pak® Light),which was pre-rinsed with anhydrous MeCN (6 mL). More anhydrous MeCN(2×0.5 mL) was used to rinse the reaction vessel and thealumina-containing cartridge. The collected MeCN eluent was diluted withice-cold aqueous solution of 0.1 M ammonium acetate (NH₄OAc) and 0.02 Mascorbic acid (1.5 mL), then injected into semi-preparative HPLC column(Waters Symmetry, PrepC18 7μ, 7.8×300 mm). The HPLC column was elutedwith 50% MeCN in an aqueous solution of 0.1 M NH₄OAc and 0.02 M ascorbicacid at a flow rate of 5.0 mL/min. The effluent from the HPLC column wasmonitored with a UV detector (λ=440 nm) followed by a gamma radioactivedetector. The HPLC fraction containing chemically and radiochemicallypure [F-18]FDDNP product that eluted with a retention time of ˜21 minwas collected for 1.25 min. The collected HPLC fraction was diluted withwater (9 mL) then passed through a solid-phase extraction cartridge(e.g., tC18 1 cc vac Sep-Pak® (50 mg)). The extraction cartridge waswashed with sterile water (20 mL). The [F-18]FDDNP was then eluted offthe solid-phase extraction cartridge with ethanol (EtOH (0.5 mL)) into aglass vessel and mixed with saline (total 5.5 mL) and 25% human serumalbumin (total 4 mL). The pure [F-18]FDDNP product in EtOH/saline/humanserum albumin was sterilized by passing through a Millex® GV filter(0.22 μm) and collected in a sterile multi-dose vial.

In one embodiment, a method of manufacturing2-(1-{6-[(2-[F-18]fluoroethyl)(methyl)amino]-2-naphthyl}ethylidene)-malononitrile([F-18]FDDNP) includes trapping [F-18]fluoride ion from a [F-18]fluorideion-containing solution in a resin cartridge. The [F-18]fluoride ion isthen eluted into a reaction vessel having a cryptand solution containedtherein by passing a potassium salt solution (e.g., K₂CO₃). The[F-18]fluoride/cryptand complex thus formed is subjected to multiplerounds of azeotropic evaporation with anhydrous acetonitrile to formdried [F-18]fluoride ion/cryptand complex residue in the reactionvessel. The dried [F-18]fluoride ion/cryptand complex is reacted withtosyloxy precursor2-{[6-(2,2-dicyano-1-methylvinyl)-2-naphthyl](methyl)amino}ethyl-4-methylbenzenesulfonate(DDNPTs) in anhydrous acetonitrile to form a reaction product. Thereaction product is then passed or flowed through an alumina cartridgeand into an injection vessel. The reaction product contained in theinjection vessel is diluted with an ice-cooled NH₄OAc/L-ascorbic acidsolution and then passed or flowed to an HPLC column. A fractioncontaining [F-18]FDDNP is collected from the HPLC column in a dilutionvessel. The collected [F-18]FDDNP contained in the dilution vessel isthen diluted with water and passed or flowed through a solid-phaseextraction cartridge followed by elution of the [F-18]FDDNP withethanol. The eluted [F-18]FDDNP (with ethanol) is then diluted withsaline and human serum albumin to form a final product (i.e.,formulation).

The method is used to synthesize [F-18]FDDNP ready for humanadministration with high yields and short production times. The methodalso does not use a combination of hazardous organic solvents that havebeen used in prior synthesis operations. For example, there is nomethanol, dichloromethane, or tetrahydrofuran in the final product. Tothe extent that the final product contains trace amounts ofacetonitrile, the amount is well below FDA guideline levels.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates the reaction that is used to produce[F-18]FDDNP.

FIGS. 2A-2B illustrate a schematic diagram of the semi-automatedsynthesis module used for the synthesis of [F-18]FDDNP; including unitoperations performed by various components of the module.

FIG. 3A illustrates the radiation trace illustrating the elution of[F-18]FDDNP from a separate analytical HPLC column. Note that theelution of [F-18]FDDNP is done using a quality control setup thatutilizes a different HPLC column as well as different mobile phaseconditions which causes the elution at around 9.5 minutes which is muchsooner than the elution time through the semi-preparative HPLC column.

FIG. 3B illustrates the ultra-violet (UV) light absorption traceillustrating the elution of [F-18]FDDNP from the HPLC column of FIG. 3A.

DETAILED DESCRIPTION OF ILLUSTRATED EMBODIMENTS

FIG. 1 illustrates the radiochemical reaction that leads to theproduction of the biomarker or PET tracer2-(1-{6-[(2-[F-18]Fluoroethyl)(methyl)amino]-2-naphthyl}ethylidene)malononitrile([F-18]FDDNP) from the tosyloxy precursor2-{[6-(2,2-dicyano-1-methylvinyl)-2-naphthyl](methyl)amino}ethyl-4-methylbenzenesulfonate(DDNPTs). Subsequent to preparation or synthesis, the PET tracer[F-18]FDDNP is purified by semi-preparative High Performance LiquidChromatography (HPLC). FIGS. 2A-2B illustrate a schematic diagram of thesemi-automated module 10 used for the preparation of [F-18]FDDNP. Thesemi-automated module 10 is setup in a “hot cell” that is found in acyclotron or other radiochemistry laboratory. The hot cell is adedicated enclosure that is used for the handling of radioactivematerials. The hot cell is exhausted and radiation shielded enclosuresthat prevent the technician or operator from exposure to radiation fromgamma ray emitters. Various types of concrete, lead, lead glass, steel,and depleted uranium can be used for shielding materials.

As explained herein, some components of the semi-automated module 10 maybe located outside the hot cell. These may include the dose calibratorreadout 12, heater controller 14, HPLC pump 18, HPLC solvent reservoir19, and strip chart recorder or plotter 20, as well as input lines ortubing that are used to delivery various reagents and solvents to themodule 10. The heating bath 22, valves (V1-V5), HPLC injector 24,detectors 26, 28, stirring machine 30 are remotely controlled via powerswitches (not illustrated) located outside the hot cell.

As seen in FIGS. 2A-2B, the semi-automated module 10 has five (5)functional units (i.e., identified as UNITS 1-5), with each unitperforming one of the five steps of the radiosynthesis (fluorination,pre-purification, separation, product extraction, formulation). Itshould be appreciated that UNITS 1-5 are not physically separate units;a single semi-automated module 10 is used for the synthesis. The UNITS1-5 that are described herein are referred to as individual units thateach performs different operations or processes in the synthesis andfinal formulation yet are part of a single overall module 10. Theindividual components of the units are connected to one another viatubing or lines (e.g., Polytetrafluoroethylene (PTFE) or Teflon® tubing)and the terminals of the tubing may be fitted with male or female Luertips for connection with stopcocks/syringes. During the operation,reagents and solvents are added to the system remotely via tubing orlines (e.g., Line 1-Line 10 as seen in FIGS. 2A and 2B) by applicationof either vacuum or positive pressure from outside of the hot cell,using a 60 mL syringe, nitrogen gas (for applying positive pressure), orother source of vacuum.

Described below are the individual units and their construction andfunction.

UNIT-1 Fluorination unit: This unit includes an automated pinch valve(V1) with a loop or flow path that is used for delivering [F-18]fluorideion to a resin cartridge 42 for subsequent elution. In one embodiment,the resin cartridge 42 is an anion exchange resin cartridge that isprepared to isolate [F-18]fluoride ion from proton irradiated[O-18]water in order to conserve the [O-18]water and reduce the amountof water that needs to be evaporated during the synthesis. In oneembodiment the resin cartridge 42 is custom made by loading 0.125 inch(inner diameter), 0.160 inch (outer diameter) PTFE tubing with BioRad®MP-1 resin (catalog #1411851 available from BioRad®, Hercules, Calif.).The BioRad® MP-1 resin is a macroporous strong anion exchange resin thatuses a matrix of styrene divinylbenzene. The functional group isR—CH₂N⁺(CH₃)₃. The BioRad® MP-1 resin is packed into the tubing usingeither gravity settling or pulling the resin in the tube with vacuum.The resin is retained in the tubing using a pair of frits (made from 70μm pore polyethylene frit material) that are formed with a ⅛ inch holepunch. The frits secure the resin material inside the tube and permitthe passage of fluid through the resin material. The ends of the tubingmay be fitted with Luer fittings to finalize the formation of the resincartridge 42.

When V1 is off (normally open), the valve V1 pinches or closes the flowpath on the right side of the valve V1 in FIG. 2A and allows the targetwater to flow through resin cartridge 42 (also referred to as cartridgeMP-1) where the [F-18]fluoride ion is loaded onto the resin contained inthe resin cartridge 42 while the [O-18]water proceeds to the collectionvial 34. When V1 is on, the valve V1 pinches or closes the flow path onthe left side of the valve V1, whereby a potassium carbonate (or otherpotassium salt) solution can be flowed through the resin cartridge 42 toelute the [F-18]fluoride ion to the reaction vessel 44.

The anion exchange resin cartridge 42 (e.g., MP-1 cartridge) that islocated in the flow loop associated with valve V1 is for trapping[F-18]fluoride ion from the cyclotron irradiated target water. Thecartridge 42 traps [F-18]fluoride ion and lets the [O-18]water passthrough in order to be recovered in a 30 mL glass vial 34. A dosecalibrator 45 (Model CRC-15R, Capintec, Inc., Florham, N.J.) is used formeasuring the radioactivity trapped in the resin cartridge 42. The dosecalibrator 45 is a well that is located in the hot cell and holds thevalve V1 and the resin cartridge 42. The radioactivity is read bypressing the F-18 key on the instrument readout unit 12 that is coupledthe dose calibrator sensor 45. The [F-18]fluoride ion that is trapped onthe resin cartridge 42 is released by the addition of 0.4 mL of 0.25%potassium carbonate solution in water (other potassium salts may also beused), which is added via line 1 (made from PTFE), and collected in thefluorination reaction vessel 44 via the delivery line 46 (also made fromPTFE). A heating oil bath 22 sits on a motorized jack platform (notshown) that permits the bath to selectively contact with reaction vessel44. The glass fluorination reaction vessel 44 is positioned above thebath 22 with a silicon stopper 48 carrying three PTFE tubes (tube 46,line 2, and tube 50) to the reaction vessel 44. Line 2 is used for theaddition of acetonitrile for the azeotropic drying of the aqueous[F-18]fluoride ion. Line 2 is also used to add the cryptand solution(e.g., Kryptofix®/MeCN solution) as well as the precursor reagent thatcontains DDNPTs to the reaction vessel 44 that contains the dried[F-18]fluoride ion/cryptand complex residue. During the evaporation ofacetonitrile from the fluorination reaction vessel 44, a gentle gasbubbling is enabled by connecting a source of nitrogen to the PTFE line3 in UNIT 2 which transfers nitrogen via tube 50.

UNIT-2 Alumina Sep-Pak® pre-purification unit: The fluorination reactionmixture from UNIT-1 is transferred to a glass syringe barrel 52 (3 mL)coupled to an alumina-containing cartridge 54 (Sep-Pak® Alumina N PlusLight Cartridge, product number WAT023561 Waters Corporation, MilfordMass.) by applying vacuum through line 3 which draws the reactionmixture from reaction vessel 44 to the glass syringe barrel 52 via PTFEtube 50. The alumina cartridge 54 is connected to the HPLC injectionvessel 56 via PTFE tubing 58. Fluid passage through the aluminacartridge 54 is achieved by application of positive pressure (e.g., ofnitrogen) through line 3 into glass syringe barrel 52.

UNIT-3 Semi-preparative HPLC unit: The crude product mixture in theinjection vessel 56 is then transferred to an electrically actuated HPLCinjector valve 24 via line or tubing 60 that has a 3 mL loop volume byapplying vacuum through a connected PTFE line (e.g., line 5). PTFE Line4 is used for adding aqueous solution to the HPLC injection vessel 56 asseen in UNIT-2. Upon injection of the crude reaction mixture into theHPLC column 62 (Waters Symmetry PrepC18, 7μ, 7.8×300 mm) using HPLCvalve 24, the column effluent passes through an UV detector 26 followedby a gamma radioactivity detector 28. The detector signals are recordedwith a strip chart recorder 20. The three-way solenoid valve V2 permitsone to divert HPLC eluent to either waste 64 or to a collect positionwhereby the eluent enters tubing 66 for transfer to UNIT-4 (seen in FIG.2B). Note that for valves V2, V3, and V4, “NO” stands for normally open.NC stands for normally closed.

UNIT-4 Product extraction unit: The HPLC fraction from UNIT-3 isreceived in a glass dilution vessel 68 via tubing 66 and is capped witha septum 70 carrying multiple lines. PTFE line 6 is used for dilution ofthe HPLC fraction with water, a transfer line 72 is used to siphon thecontent of the vessel 68 through a three-way solenoid valve V3, and line7 acts as a vent line. The contents of the dilution vessel 68 aremagnetically stirred with stirring device 30 and a magnetic stir-bar 74.The stir-bar 74 is placed in the dilution vessel 68 in advance of thepreparation to homogenize the liquids contained therein. A cartridge 76containing a silica-based bonded phase with strong hydrophobicity(Sep-Pak® tC18 1 cc Vac Cartridge, product number WAT0549 WatersCorporation, Milford Mass.) is connected to an output of solenoid valveV3. The contents of dilution vessel 68 are flushed through the cartridge76 by applying pressure through line 6 (while line 7 is capped) to trapthe drug substance on the cartridge 76 while solvents end up in a wastevial 64 through a three-way solenoid valve V4 via line 78. When thecartridge 76 is rinsed with water via a PTFE tubing line 8, the rinsingalso ends up in the waste vial 64. When the radioactive product trappedin the cartridge 76 is released with the addition of ethanol (also inputfrom PTFE tubing line 8), the product passes through the solenoid valveV4 and is collected in a mixing vessel 80 via tubing or line 82. Theproduct in ethanol in the mixing vessel 80 is then diluted with normalsaline (total 5.5 mL) and human serum albumin (HSA) (total 4.0 mL) addedvia PTFE line 9.

UNIT-5 Final drug sterilization unit: The final drug product vial 84 iscoupled to a 25 mm sterile filter 86 (for sterilization of the productsolution) and a 4 mm sterile filter 88 (for vent) and a sterileneedle/sterile syringe 90 is assembled in advance under asepticconditions in a laminar flow hood. The contents of the mixing vessel 80from UNIT-4 are transferred through a two-way solenoid valve V5 via line92 to a glass syringe barrel 96 (10 mL) by applying vacuum through aconnected PTFE line 10. After transfer, application of positive pressurethrough line 10 pushes the product through a check valve 98 and thesterilizing filter 86 into the sterile final drug product vial 84.

In order to setup the semi-automated reaction module the followingoperations are performed:

-   -   A) All the vessels, glassware, tubing parts, and valves are        cleaned and dried before assembly.    -   B) Regenerate the semi-preparative HPLC column 62 by eluting the        column inversely with 180 mL of 80% methanol aqueous solution.    -   C) The hot cell surface area is cleaned with 70% alcohol.    -   D) Turn on the power for the module 10 and other equipment.    -   E) Turn on and set the heater controller 14 for the oil bath 22        (front panel; 40 volt input) for 115° C.; check the oil bath        temperature with a digital thermometer.    -   F) After re-equilibrating the HPLC column 62 with 200 mL of the        mobile phase, check the semi-prep HPLC system. Measure the pump        flow rate by collecting mobile phase via the ‘collection line’        in a 10 mL gradual cylinder at 5 mL/min for 2 min. Note the        pressure at that flow rate, then lower the flow rate to 1 mL/min        and switch V2 to ‘waste’.    -   G) Rinse the alumina cartridge 54 with 6 mL of anhydrous MeCN        with a 6 mL syringe. H) Cool the 0.1 M NH₄OAc/0.02 M L-ascorbic        acid solution in refrigerator.    -   I) Activate the MP-1 anion resin cartridge 42 by washing with 12        mL of 1M KHCO₃ solution followed by 2×12 mL of 18MΩ water.    -   J) Insert the activated MP-1 resin cartridge 42 in the pinch        valve (V1) loop and place the fluoride trap into the Capintec        well 45.    -   K) Add Kryptofix®/MeCN solution (1 mL, plastic syringe) to the        reaction vessel 44.    -   L) The product vial 84 with two sterile filters (one for        filtration 86 and one for venting 88) is ordered from Cyclotron        and assembled by Cyclotron staff in sterile environment.    -   M) Label pre-assembled 30 mL sterile product vial 84 with label        ‘18FDDNP HSA/saline/EtOH’ and batch number ‘MM-DD-YY’.    -   N) Add stirring bar 74 and 9 mL 18 MΩ water into the HPLC        fraction dilution vessel 68.    -   O) Assemble the module 10, leaving the ten (10) PTFE tubing        terminals hanging out on the door of the hot cell.    -   P) Add 2.5 mL of saline to the open mixing vessel 80 with a 6 mL        plastic syringe.    -   Q) Fill the HPLC injection loop 24 with HPLC mobile phase via        line 5.

A number of solutions are prepared prior to the reaction process. Thisincludes: (1) Kryptofix®/MeCN solution (10 mg/mL); (2) KHCO₃ solution(1M); (3) K₂CO₃ solution (0.25% weight:volume basis); (4)NH₄OAc/L-ascorbic acid solution (0.1 M/0.02 M); (5) HPLC mobile phase(MeCN/0.1 M NH₄OAc+0.02 M L-ascorbic acid).

[F-18]Fluorination Reaction:

(1) Delivery of [F-18]fluoride ion solution (typically 1 to 5 Ci basedon cyclotron production capabilities) to the reaction vessel 44:

-   -   a. Note down the radioactivity (& time) of [F-18]fluoride ion        trapped on anion resin cartridge 42 using readout 12.    -   b. Turn on valve V1 (depress the electrical switch on the hot        cell panel).    -   c. Release the [F-18]fluoride ion to the reaction vessel 44,        pre-loaded with 1 mL of Kryptofix® solution, by passing 0.4 mL        of 0.25% K₂CO₃ solution followed by 0.1 mL 18 MΩ water through        the resin cartridge via Line 1.    -   d. Turn off V1 (elevate the electrical switch on the hot cell        panel).    -   e. Note down the radioactivity (& time) of [F-18]fluoride ion        residue in using readout 12.    -   f. Open the side door of the hot cell and quickly remove the        F-18 delivery line from the reaction vessel.

(2) Drying [F-18]fluoride/Kryptofix® complex:

-   -   a. Introduce a gentle stream of nitrogen to the [F-18]fluoride        ion solution via line 3 by adjusting the nitrogen flow rate with        a metering valve.    -   b. Raise the jack that holds to oil bath 22 to immerse the        reaction vessel 44 in the oil bath 22 heated to 115° C.    -   c. When the ground glass joint at the top of the reaction vessel        44 appears partially dry (˜5-6 min), add 0.5 mL of anhydrous        acetonitrile via line 2 to the reaction vessel 44 and continue        with nitrogen bubbling.    -   d. Repeat twice more the azeotropic evaporation with anhydrous        acetonitrile with 0.5 mL each time. At this point, the ground        glass joint of the reaction vessel 44 should appear completely        dry (˜15 min in total).    -   e. Decrease the temperature of the oil bath 22 from 115° C. to        93° C. during the last two evaporations of acetonitrile.

(3) [F-18]Fluorination reaction:

-   -   a. Lower the oil bath 22 and add precursor DDNPTs (2.2-2.5 mg)        in anhydrous acetonitrile (0.7 mL) with a 1 mL glass syringe via        line 2 to the dried [F-18]fluoride ion/Kryptofix® complex        residue in reaction vessel. Details regarding the synthesis of        DDNPTs may be found in Liu et al., High-Yield, Automated        Radiosynthesis of        2-(1-{6-[(2-[18F]Fluoroethyl)(methyl)amino]-2-naphthyl}ethylidene)malononitrite        ([18F]FDDNP) Ready for Animal or Human Administration, Molecular        Imaging and Biology, Vol. 9. pp. 6-16 (2007), which is        incorporated by reference herein.

Mix the contents of reaction vessel by gentle bubbling of nitrogen for afew seconds.

-   -   b. Elevate the oil bath 22 to heat the reaction mixture at        90-95° C.    -   c. Stop heating of the reaction vessel 44 at fifteen (15)        minutes time point by lowering the oil bath 22.    -   d. Cool the reaction mixture by bubbling nitrogen stream for 2        minutes.

Pre-Purification:

(1) Transfer the cooled reaction mixture from the reaction vessel 44 tothe syringe barrel 52 by pulling vacuum with a 60 mL plastic syringe vialine 3.

(2) Push with air pressure via line 3 the liquid through the aluminaSep-Pak® cartridge 54 and into the injection vessel 56.

(3) Add 0.5 mL of anhydrous MeCN to the reaction vessel 44 and repeatsteps 1 and 2.

(4) Add another 0.5 mL of anhydrous MeCN to the reaction vessel 44 andrepeat steps 1 and 2 again.

HPLC Purification:

(1) Set the HPLC pump 18 flow rate to 5 mL/min during the cooling, e.g.operation in ¶ [0046-47] to pump MeCN/0.1M NH₄OAc+0.02M L-ascorbic acid(1:1) as prep HPLC mobile phase.

(2) Add 1.5 mL of ice-cooled/chilled NH₄OAc/L-ascorbic acid (0.1M/0.02M) solution (use 3 mL plastic syringe) via line 4 to the HPLCinjection vessel 56.

(3) Transfer the mixture in the HPLC injection vessel 56 to the loop ofHPLC injector 24 via line 60 by applying vacuum through line 5 (withdrawthe syringe attached which is also used to fill the injection loop 24with HPLC mobile phase).

(4) Inject by switching the control box outside the hot cell to“injection”.

(5) Start the strip chart recorder or plotter 20 and a stopwatch.

(6) Monitor HPLC pump 18 back pressure for potential clogging.

(7) Turn on the stirring device 30 for the dilution vessel 68 containing9 mL of 18 MΩ water.

(8) Monitor the UV and radioactive traces on the strip chartrecorder/plotter 20 and collect the radioactive peak into the dilutionvessel 68 (by turning on V2) at ˜21 min retention time as observed onthe strip chart recorder/plotter 20. Stop the collection after 1 minuteand 15 second (by turning off V2).

Extraction of Drug Substance from HPLC Fraction:

(1) Continue to stir for another 1 min.

(2) Pass the mixed liquid in the dilution vessel 68 through thecartridge 76 and let the effluent go into the HPLC waste flask 64 byapplying pressure via line 6. Plug the vent tubing line 7 with astopcock, if higher pressure is needed to pass the solution through thecartridge 76.

(3) Turn on V3 and pass 12+8 mL of sterile water (use 12 mL plasticsyringe) via line 8 to wash the cartridge 76. Let the eluent go into thewaste 64.

(4) Dry the cartridge 76 with a nitrogen stream.

Drug Dose Preparation and Sampling for QC:

(1) Turn on ‘product 3-way valve’ V4 to ‘collection’.

(2) Elute the cartridge 76 with 0.5 mL of EtOH (use 1 mL sterilesyringe) via line 8 slowly into the mixing vessel 80 containing 2.5 mLof sterile normal saline.

(3) Gently bubble nitrogen into mixing vessel 80 via line 8.

(4) Stop the bubbling.

(5) Take ˜100 μL of the 16.7% EtOH/saline solution (Sample-1) fromvessel 80 for QC.

(6) Add 3 mL of 25% human serum albumin (HSA) via line 9 to the mixingvessel 80 with very gentle bubbling of nitrogen.

(7) Stop nitrogen bubbling, turn on V5 and transfer the HSA/EtOH/salinesolution by applying vacuum via line 10 to the HSA Syringe barrel 96.

(8) Turn off V5 and apply pressure via line 10 to push the productthrough the sterile filter 86 into the sterile product vial 84.

(9) Add another 1 mL of 25% human serum albumin and 3 mL of sterilenormal saline via line 9 to the residue in mixing vessel 80.

(10) Turn on V5 and transfer the rinsing HSA by applying vacuum via line10 to the syringe barrel 96.

(11) Turn off V5 and apply pressure via line 10 to transfer HSA throughthe sterile filter 86 into the product vial 84.

(12) Check filter integrity.

(13) Take 0.3 mL sample of the drug product (Sample-2) for bacterialendotoxin and sterility QC tests

Power Down

(1) Turn off the power for the whole module 10 inside the hot cell. Turnoff the HPLC pump 18 around 25 min time point. Turn off the power strip20 for the instruments outside of the hot cell.

The radiochemical yield (%) that is produced using this method is high;generally above 35% as seen below in Table 1. The radiochemical yield iscalculated from the radioactivity of the product corrected toEOB÷Radioactivity delivered to the reaction vessel 44 corrected toEOB×100.

TABLE 1 Typical radiochemical yields of finally formulated [F-18]FDDNPwith the method described herein Starting [F-18]⁻ activity [F-18]FDDNPYield Radiochemical (mCi corrected to EOB) (mCi corrected to EOB) Yield(%) 1138.5 440.9 38.7 1125.3 480.8 42.7 1242.6 457.0 36.8 1078.7 426.639.5 1133.4 529.1 46.7 1016.6 440.4 43.3 1160.6 486.3 41.2 EOB = End ofbombardment for the cyclotron production of [F-18]fluoride ion.

FIG. 3A illustrates the radiation trace illustrating the elution of[F-18]FDDNP from a separate analytical HPLC column. Note that theelution of [F-18]FDDNP is done using a quality control setup using adifferent HPLC column (i.e., an analytical column) as well as differentmobile phase conditions which causes the elution at around 9.5 minutes.FIG. 3B illustrates the ultra-violet (UV) light absorption traceillustrating the elution of [F-18]FDDNP from the HPLC column of FIG. 3A.For quality control purposes, radio thin layer chromatography (TLC) andgas chromatography tests may also be performed.

There are several important advantages of the method of manufacturing[F-18]FDDNP disclosed herein. First, the method does not incorporate acombination of hazardous organic solvents. Prior methods of synthesisrequired the use of dichloromethane (DCM), methanol (MeOH), andtetrahydrofuran (THF). See e.g., Liu et al. (2007), discussed supra.These organic solvents are classified as FDA Class 2 solvents and, undercurrent FDA regulations and guidance, should be limited inpharmaceutical products because of their inherent toxicity. For example,the FDA has issued guidance for industry Q3C Impurities: ResidualSolvents, which makes recommendations as to what amounts of residualsolvent are considered safe in pharmaceuticals. See Q3C Tables and ListGuidance for Industry, U.S. Department of Health and Human Services,Food and Drug Administration, Revision 3, June 2017, which isincorporated by reference herein. Solvents such as DCM and THF are notdesirable to include in pharmaceutical products. For example, DCM is aknown carcinogen. THF is a peroxide forming compound that is a knownirritant to body tissues. Thus, in one embodiment, the manufacturingprocess is substantially free of organic solvents such as DCM, MeOH, orTHF. The final product as described is ready for human administrationunder existing FDA regulations and guidance since the final productcontains only a trace amount of acetonitrile (0-50 ppm) which is wellbelow FDA established guideline limits (410 ppm). While the finalproduct may contain some ethanol, the amount of ethanol is diluted forhuman administration to bring the concentration well below FDA guidelinelimits. Another benefit of the current method of manufacturing is thefinal product can be produced in less elapsed time (e.g., 100 minutestotal as compared to 120 minutes). This is enabled by using a singlealumina cartridge (i.e., Sep-Pak®) to substitute for the prior synthesismethod that used 1% HCl aqueous solution to quench the reaction followedby C-18 Sep-Pak® extraction with an organic solvent and subsequentevaporation of the eluent. See e.g., Liu et al. (2007), discussed supra.Importantly, this is a non-aqueous workup that uses a single aluminacartridge directly after the fluorination reaction for pre-purification.This plays a significant role in reducing the autoradiolysis of[F-18]FDDNP in addition to shortening synthesis time. Thus, the methodcan produce a final product in less than 120 minutes in some embodimentsand, more preferably, produce a final product in 100 minutes or less.For example, in one embodiment, the final product may be produced inabout 90 to 100 minutes. This is significant because rapid radioactivedecay that begins to occur as soon as the radioactive fluorine isotopeis created (the half-life of fluorine-18 is 110 minutes). Anotheradvantage is the high yields that are achieved using the methoddescribed herein. Generally, final product yields (when measured at thefinal, formulated product) range between about 30% to about 40%.However, yields higher than this have been obtained with the highestyields being around 45-46%. The method described herein is highlyreliable and can produce up to one hundred (100) 10 mCi batch doses ofpure, high specific activity (typically from 1 to 5 Ci/micromole)[F-18]FDDNP biomarker ready for human injection using availablebiomedical cyclotrons routinely producing up to 5-10 Ci of[F-18]fluoride ion.

While embodiments of the present invention have been shown anddescribed, various modifications may be made without departing from thescope of the present invention. The invention, therefore, should not belimited, except to the following claims, and their equivalents.

1-15. (canceled)
 16. A product comprising2-(1-{6-[(2-[F-18]fluoroethyl)(methyl)amino]-2-naphthyl}ethylidene)-malononitrile([F-18]FDDNP) that is substantially free of acetonitrile formed by theprocess of: trapping [F-18]fluoride ion in a resin cartridge; elutingthe [F-18]fluoride ion into a reaction vessel having a cryptand solutioncontained therein by passing a potassium salt solution followed by waterthrough the resin cartridge, wherein a [F-18]fluoride/cryptand complexis formed therein; subjecting the [F-18]fluoride/cryptand complex tomultiple rounds of azeotropic evaporation with anhydrous acetonitrile toform dried [F-18]fluoride ion/cryptand complex residue in the reactionvessel; reacting the dried [F-18]fluoride ion/cryptand complex withtosyloxy precursor2-{[6-(2,2-dicyano-1-methylvinyl)-2-naphthyl](methyl)amino}ethyl-4-methylbenzenesulfonate(DDNPTs) in anhydrous acetonitrile to form a reaction product; passingthe reaction product through an alumina cartridge and into an injectionvessel; injecting the reaction product contained in the injection vesselto an HPLC column; collecting a fraction containing [F-18]FDDNP from theHPLC column in a dilution vessel; diluting the collected [F-18]FDDNPcontained in the dilution vessel with water; and wherein the finalproduct contains trace amounts of acetonitrile (0-50 ppm) at aconcentration below FDA established guidelines (410 ppm).
 17. Theproduct of claim 16, wherein the final product is contained in a sterilevial.
 18. The product of claim 17, wherein the final product is filteredwith a filter prior to being disposed in the sterile vial.
 19. Theproduct of claim 18, wherein the final product residue is rinsed throughthe filter with saline and human serum albumin into the sterile vial.20. The product of claim 17, wherein the [F-18]FDDNP in the sterile vialhas a radiochemical yield of greater than 35%.
 21. The product of claim17, wherein the [F-18]FDDNP in the sterile vial is produced in more than400 mCi (corrected to EOB) amounts with a radiochemical yield of greaterthan 35% with one Ci of F-18 fluoride as starting cyclotron producedactivity.
 22. The product of claim 16, wherein passing the reactionproduct directly through the alumina cartridge and into an injectionvessel further comprises rinsing the reaction vessel with anhydrousacetonitrile and flowing the rinse through the alumina cartridge. 23.The product of claim 16, further comprising adding chilled ammoniumacetate (NH₄OAc)/L-ascorbic acid to the injection vessel after passingthe reaction product through an alumina cartridge and into an injectionvessel.
 24. The product of claim 16, wherein the HPLC column is preparedwith MeCN/ammonium acetate (NH₄OAc) and L-ascorbic acid.
 25. The productof claim 16, wherein the final product is produced in less than 120minutes.
 26. The product of claim 16, wherein the final product isproduced in about 100 minutes or less.
 27. The product of claim 16,wherein the [F-18]FDDNP is produced using an automated synthesizer. 28.The product of claim 16, wherein the [F-18]FDDNP is produced using atleast some manual operations.
 29. The product of claim 16, wherein thefinal product is free of methanol, dichloromethane, and/ortetrahydrofuran.